6-Phosphofructokinase

The enzyme phosphofructokinase, isolated from a number of different sources including skeletal muscle, heart muscle, and erythrocytes, is known to undergo a concentration dependent self-association which is sensitive to pH as well as to added substrates and effector molecules. Various oligomers of phosphofructokinase have been observed to differ in specific activities and substrate affinities, suggesting that the self-association may be involved in regulation of the enzyme (ref. 13). 

Large zone SEC has been applied to the study of the self-association of human erythrocyte phosphofructokinase at pH 8, 4°C, in the presence of the substrate fructose-6-phosphate (ref. 13). Phosphofructokinase is known to 

In 1909 it was observed that a stable, phosphorylated form of fructose, fructose-1,6-diphosphate, accumulated when glucose was fermented by cell-free preparations of yeast. In 1936 it was shown that a specific enzyme was responsible for the formation of the diphosphate and could be found in yeast and muscle extracts. This enzyme, phosphofructokinase, has since been highly purified and its properties studied. This enzyme catalyzes the transfer of the terminal group of ATP to fructose-6-phos-phate in the presence of Mg++. Its specificity is high, since it does not catalyze any other transfer reaction. 

By subjecting extracts of rabbit muscle to a series of ammonium sulfate fractionations, a cut was obtained which had a turnover number of about 1400 per minute per 10 g. of protein. Since the enzyme is imusually labile to acid conditions in all purification procedures the pH had to be held above 7. There is evidence that the stability of the enzyme is affected not only by the pH but also by the nature of the ionic environment and concentration. Thus, certain anions such as oxalate, pyrophosphate, phosphate, and sulfate tend to stabilize buffered solutions of the enzyme.  

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For many years hemoglobin was the only allosteric protein whose stereochemical mechanism was understood in detail. However, more recently detailed structural information has been obtained for both the R and the T states of several enzymes as well as one genetic repressor system, the trp-repressor, described in Chapter 8. We will here examine the structural differences between the R and the T states of a key enzyme in the glycolytic pathway, phosphofructokinase. 

Figure 6.24 The function of the enzyme phosphofructokinase. (a) Phosphofructokinase is a key enzyme in the gycolytic pathway, the breakdown of glucose to pyruvate. One of the end products in this pathway, phosphoenolpyruvate, is an allosteric feedback inhibitor to this enzyme and ADP is an activator, (b) Phosphofructokinase catalyzes the phosphorylation by ATP of fructose-6-phosphate to give fructose-1,6-bisphosphate. (c) Phosphoglycolate, which has a structure similar to phosphoenolpyruvate, is also an inhibitor of the enzyme.

Figure 6.25 Schematic diagram of the structure of one dimer of phosphofructokinase. Each polypeptide chain is folded Into two domains (blue and red, and green and brown), each of which has an oi/p structure. Helices are labeled A to M and p strands 1 to 11 from the amino terminus of one polypeptide chain, and respectively from A to M and 1 to 11 for the second polypeptide chain. The binding sites of substrate and effector molecules are schematically marked In gray. The effector site of one subunit is linked to the active site of the other subunit of the dimer through the 6-F loop between helix F and strand 6. (Adapted from T. Schlrmer and P.R. Evans, Nature 343 140-145, 1990.)...

Schirmer, T., Evans, P.R. Structural basis of the allosteric behaviour of phosphofructokinase. Nature 343 140-145, 1990. 

When coupled (by phosphofructokinase) with the hydrolysis of ATP, the overall reaction (Figure 19.7) is strongly exergonic ... 

Clearly, the activity of phosphofructokinase depends both on ATP and AMP levels and is a function of the cellular energy status. Phosphofructokinase activity is increased when the energy status falls and is decreased when the energy status is high. The rate of glycolysis activity thus decreases when ATP is plentiful and increases when more ATP is needed. 

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which feeds the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated. 

FIGURE 19.9 Fructose-2,6-bisphosphate activates phosphofructokinase, iucreasiug the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate. 

As described in Chapter 19, Emile Van Schaftingen and Henri-Gery Hers demonstrated in 1980 that fructose-2,6-bisphosphate is a potent stimulator of phosphofructokinase. Cognizant of the reciprocal nature of regulation in glycolysis and gluconeogenesis. Van Schaftingen and Hers also considered the... 

If fructose-1,6-bisphosphatase and phosphofructokinase acted simultaneously, they would constitute a substrate cycle in which fructose-1,6-bisphosphate and fructose-6-phosphate became interconverted with net consumption of ATP ... 

Figure 5.3 Major control points of glycolysis and the TCA cycle. Enzymes I, hexokinase II, phosphofructokinase III, pyruvate kinase IV, pyruvate dehydrogenase V, citrate synthase VI, aconitase VII, isocitrate dehydrogenase VIII, a-oxoglutarate dehydrogenase.

Within glycolysis, the main allosteric control is exercised by phosphofructokinase, a complicated enzyme unusual in that its activity is stimulated by one of its products (ADP) and inhibited by one of its substrates (ATP). One further point about this enzyme which will be important to us later, in Aspergillus spp., elevated levels of ammonium ions relieve phosphofructokinase of inhibition by titrate. 

Nitrogen is normally supplied as an ammonium compound in dtric acid fermentations and suffident has to be supplied to enable the effect of manganese deficiency (increased levels of ammonium in the metabolic pool) to occur. Remember that increased metabolic pool ammonium has the effect of releasing the allosteric controls exerted on phosphofructokinase. 

A decreased glycolytic rate has been proposed as a cause of muscle fatigue and related to pH inhibition of glycolytic enzymes. Decreasing pH inhibits both phosphorylase kinase and phosphofructokinase (PFK) activities. PFK is rate determining for glycolytic flux and therefore must be precisely matched to the rate of ATP expenditure. The essential characteristic of PFK control is allosteric inhibition by ATP. This inhibition is increased by H and PCr (Storey and Hochachka, 1974 ... 

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